携带猪γ干扰素基因逆转录病毒载体的构建及其在猪肾细胞(PK-15)中的表达  

Construction of recombinant retroviral vector carrying porcine interferon-gamma and its expression in porcine kidney cells (PK-15)

作　　者：姚清侠[1] 刘新文[1] 钱平[1] 郭东春[1] 陈焕春[1] 

机构地区：[1]华中农业大学畜牧兽医学院动物病毒室,武汉430070

出　　处：《微生物学报》2007年第1期141-144,共4页Acta Microbiologica Sinica

基　　金：国家"863计划"(2002AA245071);湖北省科技攻关项目(2004AA202B01)~~

摘　　要：将梅山猪γ干扰素基因定向插入逆转录病毒载体pLXSN(neor),构建逆转录病毒重组质粒,利用脂质体介导法将重组质粒转染逆转录病毒包装细胞系PA317,转染细胞经含G418(400μg/mL)培养基筛选一周后获得稳定产毒的PA317细胞系。从细胞培养上清中提取RNA,进行RT-PCR检测,扩增到目的片段;将上清感染猪肾细胞(PK-15),经含G418(400μg/mL、600μg/mL和800μg/mL)的DMEM筛选一周,间接免疫荧光表明表达的猪γ干扰素主要锚定于细胞膜。收取PK-15细胞上清,在牛肾细胞(MDBK)上进行干扰素抗病毒活性检测,结果显示重组病毒表达的猪γ干扰素抗水泡性口炎病毒(VSV)的活性为1200IU/106cells.48h。以表达的干扰素处理PK-15细胞后,经细胞病变抑制法测定,重组猪γ干扰素可以抵抗口蹄疫病毒(FMDV)感染。试验结果表明猪γ干扰素基因已成功插入逆转录病毒基因组并在PK-15细胞中表达,表达的重组猪γ干扰素具有较强的抗病毒生物活性。Porcine interferon-gamma （PoIFN-γ） of Chinese local brand, Meishan porcine, was cloned and inserted into retroviral vector pLXSN （ neo ）. Using LipofectamineTM, this recombinant plasmid was transfected into retroviral packing cell line, PA317 cells. These transfected cells were selected by DMEM containing 400μg/mL G418 for one week. RNA was extracted from the supernatant of these selected PA317 cells and the PoIFN-γ gene could be amplified by RT-PCR. Pocine kidney cells and PK-15 cells were infected by the supernatant and were selected by 400μg/mL, 600μg/mL and 800μg/mL G418, respectively. Those PK-15 cells were detected by indirect immunofluorescence assay and it was found that PoIFN-γ mainly anchored in cellular membrane. The supernatant of the selected PK-15 was tested for the antiviral bioactivity after 48 hours of passage. The anti-VSV （vesicular stomatitis virus） activity in MDBK （bovine kidney cell） was 1200IU/106cells. In addition, the effect of rPoIFNγ-anti-FMDV was determined using cytopathic effect inhibition. The results indicate that PoIFN-γ has been inserted into retroviral vector and recombinant retrovirus has been successfully pack.aged in PA317 cells. Furthermore, this retrovirus can infect PK-15 cells and express PoIFN-γ with natural antiviral bioactivity and can inhibit VSV and FMDV.

关 键 词：猪Γ干扰素 逆转录病毒 PA317细胞 PK-15细胞 表达 抗病毒活性 水泡性口炎病毒 口蹄疫病毒 

分 类 号：Q78[生物学—分子生物学]